The long term objective of this project is to understand the mechanisms by which transport proteins of cell membranes function. Transport mechanisms are important in many biological processes, and they are especially important in the regulation of cell volume; which, in turn, has profound effects on cell function. For instance, in red blood cells volume regulation seems to be of great importance in modulating the severity of sickle cell disease. Understanding the mechanisms by which transport proteins function seems a prerequisite for devising strategies for influencing their function. The specific aims of this proposal are to test some hypotheses regarding three unsettled questions about the mechanism of the Na, K pump, and to examine characteristics of the volume sensitive, C1 dependent K fluxes present in red cell ghosts in order to obtain information about the way in which swelling is detected and the signal transmitted to the transport protein. We propose (1) to compare the temperature sensitivity of ATPase activity of the Na pump to the temperature sensitivity of Na-K exchange. Our goal is to decide whether measurements of partial reaction rates of 0 degree C are applicable to the pump mechanism under physiological conditions. (2) To find our whether subunits of the pump cooperate in protecting the pump against vanadate. Although it seems possible that monomers of the pump protein are capable of carrying out Na-K exchange, there are many phenomena which suggest that pump activity may at least be modulated by subunit interaction. (3) To determine whether the composition of the phospholipid membrane in which the pump is embedded, and especially its surface charge, changes the apparent affinity of the pump for its substrates. (4) To determine how ATP is utilized to support volume sensitive fluxes, and to examine the effect of manipulations which modify the strength of interactions between red cell membranes and the cytoskeleton on the volume sensitive fluxes.